Mast cells play an important role in many inflammatory and immunological reactions by releasing an array of mediators. The goal of our studies is to understand the intracellular signal transduction pathways that lead to the release of these molecules. In previous studies, we demonstrated that the protein tyrosine kinase Syk is essential for the immune receptor-induced degranulation that results in the release of inflammatory mediators. A variant of the rat basophilic RBL-2H3 mast cells that has no detectable Syk was also identified and has been used to examine the structural basis of the regulation of Syk after immune receptor aggregation. These studies identified the linker region of Syk, located between the second SH2 and the kinase domain, as important in regulating the function of this kinase. To identify novel substrates of the protein tyrosine kinases Lyn and Syk that are critical in signaling, we screened a cDNA expression library prepared from RBL-2H3 cells for proteins that were tyrosine phosphorylated in vitro. Five clones as potential Lyn substrates and eight clones as Syk substrates were identified including known molecules. Experiments are continuing to elucidate the functional importance of several new molecules that we identified. To investigate the role of phospholipase D (PLD) in immune receptor signaling, the wild type or the catalytically inactive forms of PLD1 or PLD2 were stably overexpressed in RBL-2H3 cells. This receptor-induced PLD activation required the protein tyrosine kinase Syk and resulted in the activation of both PLD1 and PLD2. However, PLD1 was the source of most of the receptor-induced PLD activity. There was enhanced receptor-induced degranulation only in cells that overexpressed the catalytically inactive PLD1. This was found to correlate with the constitutive basal PLD1 activity that regulates phosphatidic acid formation that then controls the early signals initiated by immune receptor aggregation. The Cbl family proteins negatively regulate signaling from tyrosine kinase-coupled receptors. To examine the role of c-Cbl and Cbl-b in immunoglobulin receptor signaling, mast cell cultures were generated from wild-type, c-Cbl and Cbl-b deficient mice. Compared to control cells, Cbl-b inactivation resulted in increases in immunoglobulin receptor-induced signaling and release of inflammatory mediators. In contrast to Cbl-b, c-Cbl deficiency had no detectable effect on receptor-induced degranulation or signal transduction. These results indicate that Cbl-b and c-Cbl have divergent effects on immune receptor signal transduction and that Cbl-b, but not c-Cbl, functions as a negative regulator of degranulation. A committed mast cell precursor has long been assumed to be present in bone marrow, although its identification and isolation has proven to be difficult. We have used sequential immunomagnetic isolation with two mast cell specific antibodies to purify and characterize a lineage committed mast cell precursor from adult mouse bone marrow that represents only 0.02% of the total cells. We have studied the in vitro growth requirements and morphology of these cells and we are now characterizing the molecules that are uniquely expressed in the immature precursors.